Limnology and Freshwater Biology 2018 (2): 107-112 DOI:10.31951/2658-3518-2018-A-2-107 Original Article The diversity of hydras (Cnidaria: Hydridae) in the Baikal region

Peretolchina T.E.*, Khanaev I.V., Kravtsova L.S.

Limnological Institute, Siberian Branch of the Russian Academy of Sciences, Ulan-Batorskaya Str., 3, Irkutsk, 664033, Russia

ABSTRACT. We have studied the fauna of Hydra in the waters of the Baikal region using morphological and molecular genetic methods. By the external morphological features of the structure of the polyp and the microscopic study of nematocysts, we have identified four species belonging to three different genetic groups: “oligactis group” (Hydra oligactis, H. oxycnida), “braueri group” (H. circumcincta) and “vulgaris group” (H. vulgaris). Molecular phylogenetic analysis has confirmed the species status of the hydras studied. The intraspecific genetic distances between the Baikal and European hydras are 1.5– 4.2% and 0.4–2.7% of the substitutions in COI and ITS1–5.8S–ITS2 markers, respectively, while, inter- specific distances for different species significantly exceed intraspecific and amount to 9.8−16.1% and 6.4−32.1% of substitutions for the markers COI and ITS1–5.8S–ITS2, respectively. The temperature regime of the water and the availability of food resources, which play a key role in the reproduction of hydras, determine the habitats of the identified species. The research performed has replenished the regional fauna with species whose findings were previously considered presumptive or doubtful. The first record of H. vulgaris in the artificial reservoir near the Angara River (Lake Kuzmikhinskoye) will help to clarify the distribution of this species.

Keywords: Baikal region, Hydra, nematocysts, molecular phylogenetic analysis

1. Introduction 2. Materials and methods

Hydra is a member of the ancient phylum Hydras were collected at depths of 1 to 18 meters Cnidaria, class Hydrozoa, order Hydroida, family by a scuba diver and with hands: 1) along the littoral Hydridae. Genus Hydra comprises 15 species (Jankowski zone of Lake Baikal; 2) in Posolsky Sor. Hydras together et al., 2008), five of them are found in Europe: Hydra with aquatic plants were also collected by hands from viridissima Pallas, 1766, H. circumcincta Schulze, 1914, lakes Zama (near Lake Baikal) and Kuzmikhinskoye H. oligactis Pallas, 1766, H. vulgaris Pallas, 1766 and H. (the artificial reservoir near the Angara River) at depths oxycnida Schulze, 1914 (Schuchert, 2010). of 0.5 m (Fig. 1). Typically, representatives of Hydra There have been no detailed studies of the were brought live to the laboratory, but some samples fauna of the cnidarians in Lake Baikal. According were fixed in 80% ethanol in the field. to available literature data, four species of fresh- The nematocysts of Hydra representatives water hydroids of the genus Hydra were recorded in (stenoteles, desmonemes holotrichous and atrichous Lake Baikal: H. baikalensis, H. oligactis and, rarely, H. isorhizas) were photographed using Olympus CX22 circumcincta, H. oxycnida (= H. oxycnidoides sensu microscope with 1000-fold magnification under Shultze, 1927) (Kozhov, 1962; Stepanyants et al., 2003; oil immersion. Morphometric measurements of 2006). Gajewskaja supposed the existence of H. vulgaris holotrichous isorhizas and stenoteles were carried out in Lake Baikal (Gajewskaja, 1933); however, subse- with the Image-Pro program (Table 1). Hydras spe- quent researchers never found H. vulgaris, as well as H. cimens were identified according to keys provided by oxycnida, and doubted their presence in the lake. In the Schuchert (2010) and Anokhin (2002). “Index of animal species inhabiting Lake Baikal and its DNA was extracted from a single live or fixed catchment area”, only two species are mentioned: H. specimen according to the protocol described by Doyle oligactis and H. baikalensis (Stepanyants and Anokhin, and Dickson (1987). Gene fragments of mitochondrial 2001). cytochrome c oxidase subunit I (COI) and internal The aim of this work was to study the diversity transcribed spacer 1 – 5.8S ribosomal DNA – internal of the fauna of the hydroids in the Baikal region using transcribed spacer 2 (ITS1–5.8S–ITS2) were amplified morphological and molecular genetic methods. in PCR using the primers listed in Table 2. The PCR

*Corresponding author. E-mail address: [email protected] (T.E. Peretolchina) © Author(s) 2018. This work is distributed under the Creative Commons Attribution 4.0 License. 107 Peretolchina T.E. et al. / Limnology and Freshwater Biology 2018 (2): 107-112

Fig. 1. A – Sampling sites of Hydra; B – photos of massively growing H. oligactis on the dying Porifera (a, b) and on the fila- mentous algae (c) (photos by I.V. Khanaev)

amplification conditions for both molecular markers rior probabilities of the phylogenetic tree, we used were as follows: denaturation at 94 ºC for 5 min, 30 15,000,000 generations of Metropolis-coupled Markov cycles at 94 ºC for 30 sec, 50 ºC for 45 sec, 72 ºC for chain Monte Carlo simulation (two runs with four 2 min, and a final elongation step at 72 ºC for 10 min. chains). We used the jModelTest v.2.1 (Darriba et al., Direct sequencing of forward sequences was performed 2012) to determine the substitution models for the three in Research and Production Company SYNTOL (Russia) genes separately. The best-fit model for phylogenetic using an ABI 3130 automated sequencer. analysis in each case was GTR+I+G. We constructed The DNA sequences obtained were edited in a majority-rule (50%) consensus tree following 25% BioEdit v.7.2.5 (Hall, 2011) and aligned using ClustalW burn-in of all sampled trees to allow likelihood values (Thompson et al., 1994) and MAFFT v.6.240 (Katoh et to reach stationary equilibrium. al., 2002). Resulting COI alignment was translated to check for the absence of stop codons. To confirm species 3. Results and discussion identity, we compared our sequence dataset with the published ortholog sequences from other members of All Hydra samples collected were identified by Hydra and analyzed genetic distances using Mega v.6 external morphology of polyps and microscopic exa- (Table 3) (Tamura et al., 2013). mination of their nematocysts as H. oligactis, H. oxycnida Phylogenetic analysis was performed using (“oligactis group”), H. circumcincta (“braueri group”) MrBayes v.3.2 (Ronquist and Huelsenbeck, 2003). and H. vulgaris (“vulgaris group”) (Fig. 2). The dataset consisted of eight combined COI+ITS1– The consensus tree topology (Fig. 3) based on 5.8S–ITS2 sequences: four sequences of hydra spe- combined sequence data (COI and ITS1–5.8S–ITS2) cimens produced for this study and four ones of spe- have confirmed that hydras from the Baikal region cimens belonging to European species: H. circumcincta belong to four species: H. circumcincta, H. oligactis, H. (GU722853, GU722669), H. oligactis (GU722868, vulgaris and H. oxycnida. The intra-specific distances GU722691), H. vulgaris (GU722918, GU722744) and of hydras from the Baikal region and Europe do not H. oxycnida (GU722876, GU722690) (Martínez et al., exceed 5% that indicate species level of the samples 2010) retrieved from GenBank. To estimate the poste- studied (Martínez et al., 2010; Schwentner and Bosch,

Table 1. The nematocysts sizes of different species of Hydra from the Baikal region

Species holotrichous isorhiza stenotele width, µm length, µm n width, µm length, µm n x±m min max x±m min max x±m min max x±m min max H. oligactis 4.2±<0.1 3.1 5.2 8.8±0.1 7.4 11.4 100 9.6±0.1 9.1 9.9 12.9±0.2 12.2 13.6 8 H. oxycnida 4.2±0.1 3.9 4.8 10.1±0.2 8.9 11.4 15 11.8±0.2 10.7 12.8 20.3±0.5 18.0 22.8 9 H. circumcincta 5.8±0.1 4.9 6.7 8.6±0.1 7.5 9.4 29 11.6±0.5 10.6 12.6 13.6±<0.1 13.5 13.6 3 H. vulgaris 4.7±0.1 4.2 5.2 10.2±0.2 7.9 12.4 19 12.6±2.2 9.9 25.6 16.4±2.6 11.6 31.9 7 x- mean, m – standard error of mean, min and max – limits, n – number of measurements

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Table 2. Primers used in this study

Gene Primer Reference COI LCO1490: 5’-GGT CAA CAA ATC ATA AAG ATA TTG G-3’ Folmer et al. (1994) HCO2198: 5’-TAA ACT TCA GGG TGA CCA AAA AAT CA-3’

ITS1–5.8S–ITS2 ITS1 (f): 5’-TCC GTA GGT GAA CCT GCG G-3’ White et al. (1990) ITS4 (r): 5’- TCC TCC GCT TAT TGA TAT GC-3

2015). Inter-specific distances varied from 9.8% to America (Hyman, 1930; Holstein, 1995; Stepanyants et 16.1% between COI sequences and from 6.4% to 32.1% al., 2006). between ITS1–5.8S–ITS2 sequences for different species Hydra oxycnida Schulze, 1914, syn.: Hydra of Hydra (Table 3). Large genetic distances between oxycnidoides Schulze, 1927, Hydra pirardi Brien, 1961. hydras belonging to different genetic groups deter- Description. Very large dark brown Hydra without mined by ITS1–5.8S–ITS2 marker can be explained by pedicel, stenoteles are markedly ovoid (Fig 2D), width/ presence of indels obtained after alignment. length ratio is 0.6, and upper part is characteristically Below are the ecological and morphological cha- pointed. Holotrichous isorhizas are ovoid to oval, the racteristics of the studied species (the description of the length/width ratio is >2 (Fig. 2B), there is shaft in species based on morphological characters according intact capsule thread in 3-4 oblique or transverse coils to Schuchert (2010) and measurements of nematocysts in the upper part of capsule. Tentacles are 7-8, rarely (Table 1)): 6-11, relatively short, extended and reaching only 1/4 Phylum Cnidaria Verrill, 1865 to 1/3 of body length, polyp buds form tentacles simul- Subphylum Medusozoa Peterson, 1979 taneously. Sexes are strictly separated, no sex change, Class Hydrozoa Owen, 1843 the onset of gametogenesis stops vegetative budding Subclass Hydroidolina Collins 2000 and nematocyst production. Order Anthoathecata Cornelius, 1992 Distribution. Lake Baikal (Posolsky Sor). Family Hydridae Dana, 1846 Additionally, it occurs in Russia (Holstein, 1995) and is Hydra oligactis Pallas, 1766, syn.: Hydra fusca common in Northern Europe (Schuchert, 2010). Linnaeus, Hydra roeselii Haacke, Hydra rhaetica Asper, Hydra circumcincta Schulze, 1914, syn.: Hydra Hydra rhistica Asper, Pelmatohydra oligactis Schulze. braueri Bedot, 1912, Hydra stellata Schulze, 1914, Hydra Description. The Baikal Hydra is large, typically ovata Boecker, 1920, Hydra graysoni. with 5–7 long tentacles and a more or less distinct Description. Small Hydra with short tentacles, no pedicel. The cnidome includes four types of nema- pedicel, holotrichous isorhizas are ovoid (Fig. 2K), the tocysts: stenoteles, holotrichous isorhizas, atrichous length/width ratio is <1.5, there is thread in 3-4 distinct isorhizas and desmonemes (Fig. 2E–H). Holotrichous coils in the upper part of capsule, stenoteles are ovoid isorhizas are elongated, with a length/width ratio of (Fig. 2L), with two size classes, the width/length ratio approximately 2, and with thread-forming longitudinal is approximately 0.85. Hypostome is conical, below irregular coils inside the capsule. Other types of nema- hypostome there are usually six tentacles, sometimes tocysts are of a size and shape typical of hydras. Sexes five tentacles, tentacles are shorter than hydranth body are strictly separated, no sex change, the onset of game- (ratio < 0.5), held horizontally. Contracted hydranths togenesis stops vegetative budding. are with star-shaped tentacle crown. Vegetative multi- Distribution. Lake Baikal: Port Baikal, Ulanovo, plication is by buds, which are relatively basal, ten- Listvennichny Bay, Bolshie Koty, Varnachka, Sobolev tacles of buds are formed simultaneously, usually six in Cape, Turali Cape, Elokhin Cape, Mukhor Bay, Posolsky number. Vegetative and sexual reproduction can occur Sor; Lake Kuzmikhinskoye (Artificial reservoir near simultaneously. Sexual reproduction is as hermaphro- the Angara River). This species is also widespread and dites, sometimes proterandric. common on the entire European continent, including Distribution. Lake Zama (near Lake Baikal). the British Isles and Iceland as well as Russia and North They are also found in Russia, Japan, North America

Table 3. Pairwise p-distances between COI (below diagonal) and ITS1–5.8S–ITS2 (above diagonal) for different species of Hydra. Intra-specific pairwise p-distances are in bold.

H. oxycnida H. oligactis H. vulgaris H. circumcincta H. oxycnida 0.015 (0.004) 0.064 0.205 0.317 H. oligactis 0.098 0.018 (0,007) 0.214 0.321 H. vulgaris 0.123 0.122 0.042 (0.026) 0.251 H. circumcincta 0.161 0.158 0.132 0.036 (0.027)

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Fig. 2. Nematocysts of different Hydra species: A–D – H. oxycnida; E–H – H. oligactis; I–L – H. circumcincta; M–O – H. vulgaris. A, E, J, N – atrichous isorhizas; C, F, I – desmonemes; B, G, K, M – holotrichous isorhizas; D, H, L, O – stenoteles. Scale bar is 10 μm.

(Heitkamp, 1986; Holstein, 1995; Stepanyants et al., 2006) and in Europe, including the British Isles, except for Iceland (Schuchert, 2010).

Hydra vulgaris Pallas, 1766, syn.: Hydra grisea Linnaeus, 1767, Hydra vulgaris aurantiaca Ehrenberg, 1838, Hydra trembleyi Haacke, 1879. Description. Medium sized Hydra, extended hydranth is usually 3-6 mm, may reach sizes of up to 15 mm, with 6-8 long tentacles, without algal symbi- onts, no or indistinct pedicel, holotrichous isorhizas are oblong, the length/width ratio is >2, thread is in 4-5 oblique or transverse coils the in upper part of capsule, stenotele width/length ratio is approximately 0.78 (Fig. 2M, O). Vegetative multiplication is by buds located in lower third of the body, usually there are Fig. 3. Combined COI and ITS1–5.8S–ITS2 phylogenetic only 1-2 buds, rarely 3-4, initial four tentacles of buds tree. Haplotypes of hydras from the Baikal region are in bold are formed more or less simultaneously, later more tentacles develop, and, thus, tentacles are transiently of unequal length. Vegetative and sexual reproduction sentative of freshwater cnidaria in almost all conti- can occur simultaneously. Sexual reproduction of gono- nental waters. Previously, representatives of hydras choristic and hermaphroditic animals is very rare. In were found in small numbers in the coastal zone of Lake hermaphrodites, male and female gonads are mixed, Baikal. The first record of the mass development of H. and are not in separate regions. oligactis was in the 2000s (Stepanyants et al., 2003). Distribution. Lake Kuzmikhinskoye (Artificial Now, it is abundant along the open coasts of all three reservoir near the Angara River). In Russia, it was basins of Lake Baikal. In the last decade, the blooming found in the Far East (Stepanyants et al., 2006). It also of filamentous algae (Spirogyra, Ulotrix and others) and inhabits the entire European continent and the British the trend towards a more widespread distribution of Isles (Schuchert, 2010). invasive species, in particular, Elodea canadensis, have Currently, among the hydras listed above, in been observed in Lake Baikal (Kravtsova et al., 2010). open Lake Baikal only H. oligactis occurs, mass growth In addition, mass death of the endemic species of of which has been observed in the last decade (Fig. Porifera (Denikina et al., 2016; Khanaev et al., 2018) is 1B). Representatives of this species are capable of currently observed. On the contrary, representatives of very intense budding: 10–20 young polyps that have Hydra acquire a significant role along the open coasts not yet budded are found on one maternal specimen of Lake Baikal. (Stepanyants et al., 2003; Tökölyi et al., 2016). For this According to the literature data, the temperature reason, H. oligactis became the most widespread repre- optimum for sexual reproduction of H. oligactis is 10–12

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°C, and for vegetative budding is 18–22 °C (Littlefield, the water bodies of Europe and the Far East. 1991). The water temperature in the surface layer of the coastal zone of the Lake Baikal in summer does not Conclusions exceed 16 °C (Timoshkin et al., 2018). Despite the fact that the water temperature in Baikal is slightly lower Thus, in the Baikal region, the fauna of hydras is than the temperature optimum for vegetative repro- represented by typical inhabitants of freshwater bodies duction, H. oligactis exhibits intensive budding and of northern latitudes. The morphological and molecular develops along the open coasts of the lake in all three genetic analysis of the hydras from the Baikal region basins. water bodies studied allowed us to uniquely identify Due to the global climate change since the early four species: H. oligactis, H. oxycnida, H. circumcincta 70s of the past century, an increase in air temperature and H. vulgaris. In open part and in the coastal zone has been observed in the Baikal region during all seasons of Lake Baikal, only representatives of the “oligactis of the year, especially in winter, as well as an increase group” were found: H. oligactis and H. oxycnida. The in the duration of the ice-free period of Lake Baikal and regional fauna of the hydra has been replenished with warming of its surface waters (in May–September) by 1 species whose findings were previously considered °C (Shimaraev et al., 2002). However, such increase in presumptive or doubtful. The record of H. vulgaris in the temperature hardly contributes to the mass distribution Baikal region makes the species area less fragmented of other Hydra species in open Baikal, in particular, H. since it was previously found only in water bodies of oxycnida, H. circumcincta and H. vulgaris. Europe and the Far East. 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